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Blast heuristics Morten Nielsen Department of Systems Biology, DTU Outline • Basic Local Alignment Search Tool – What are the Blast heuristics? – How does Blast calculate E-values? – What are the limits of Blast? • Psi-Blast – Why does it work so much better – This we saw last week ... It uses sequence profiles Why alignment is slow • 99% of the cpu time is spend aligning nosimilar sequences • The execution time for gapped alignments is 500 times that for un-gapped Blast heuristics • Hits (High scoring segment pairs, HSP) – Triplets of amino acids that scores at least T Hit extension What are P and E values? • E-value – Number of expected hits in database with score higher than match – Depends on database size • P-value – Probability that a random hit will have score higher than match – Database size independent Score 150 10 hits with higher score (E=10) 10000 hits in database => P=10/10000 = 0.001 Score Blast E-values • Normalized score S’ (bit score) is S' S ln( K) ln 2 • and K are calculable given sij and pi p p i j exp( sij ) 0 i, j • The number E of sequences with a score of at least S’ is N E S' 2 Altschul et al., 1997. Nucleic Acids Research Blast output Edge Effects • a high-scoring alignment must have some length, and therefore can not begin near to the end of either of two sequences being compared. This "edge effect" may be corrected for by calculating an "effective length" for sequences; the BLAST programs implement such a correction. Blast output Blast E-values. Example Score = 506 bits (1302), Expect = 7e-142, Identities = 245/245 (100%), Positives = 245/245 (100%) .. Lambda K 0.267 0.0410 Matrix: BLOSUM62 S' S ln( K) 1302 0.267 ln( 0.041) ln 2 ln 2 506 • The number E of sequences with a score of at N least S’ is E S' 2 log( E) log( N) S'log( 2) log(1468197536114) - 506 log(2) E 7 10142 Blast Only align subset of sequences Only do gap extension at few seed sites Only extend gaps close to diagonal Approximate (and conservative) E-value estimates • Details on the Blast algorithm • • • • – www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html